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Abstract
Objective—The goal of this study was to assess the activity of β-catenin/T-cell-specific transcription factor (TCF) signaling in atherosclerosis development and its regulation of fibronectin in vascular endothelium.
Methods and Results—Histological staining identified preferential nuclear localization of β-catenin in the endothelium of atheroprone aorta before and during lesion development. Transgenic reporter studies revealed that increased levels of TCF transcriptional activity in endothelium correlated anatomically with β-catenin nuclear localization and fibronectin deposition. Exposure of endothelial cells to human-derived atheroprone shear stress induced nuclear localization of β-catenin, transcriptional activation of TCF, and expression of fibronectin. Activation of fibronectin expression required β-catenin, TCF, and the transcriptional coactivator CRBP-binding protein. Finally, we identified platelet endothelial cell adhesion molecule-1 as a critical regulator of constitutive β-catenin and glycogen synthase kinase-3β activities.
Conclusion—These data reveal novel constitutive activation of the endothelial β-catenin/TCF signaling pathway in atherosclerosis and regulation of fibronectin through hemodynamic shear stress.
Beta-catenin (β-cat) is a highly conserved, multifunctional member of the armadillo family whose nuclear translocation and coactivation of the T-cell-specific transcription factor (TCF)/lymphoid enhancer-binding factor (LEF) family of transcription factors represents a critical step in a variety of cell processes, including development, epithelial-mesenchymal transition, angiogenesis, and differentiation.1 Studies have identified a role for TCF/LEF activity in several pathological features of advanced atherosclerotic lesions, including vascular calcification2–4 and smooth muscle cell proliferation.5 However, the involvement of this signaling pathway in the endothelium during early atherosclerosis development is poorly understood.
Cytosolic β-cat is constitutively targeted for ubiquitination-mediated degradation via glycogen synthase kinase 3β (GSK-3β)-dependent phosphorylation. On stimulation by various factors (including canonical Wnts and growth factors) GSK-3β activity is decreased, leading to nuclear accumulation of β-cat, followed by binding and activation of the TCF/LEF family of transcription factors. One target of TCF/LEF-dependent transcription is the extracellular matrix protein fibronectin.6 TCF-dependent fibronectin expression has been identified to play important roles in several cell contexts, including fibroblast differentiation,7 lung branching morphogenesis,8 and epithelial-mesenchymal transition.6 Fibronectin is also highly regulated in atherosclerotic tissue and is involved in atherosclerosis development through promotion of inflammation and endothelial permeability.9–12 However, the role of endothelial β-cat/TCF in this process remains unknown.
One prominent feature of the atherosclerotic environment is hemodynamic shear stress, which regulates the phenotype of endothelial cells (EC)13,14 and largely explains the regional bias of atherosclerosis development.15,16 Specifically, low-magnitude, reversing shear stress, such as that which occurs in branching and curved vessels,17,18 induces a chronic inflammatory phenotype in preatherosclerotic endothelium. GSK-3β inactivation occurs in response to onset of shear stress in a platelet endothelial cell adhesion molecule-1 (PECAM-1)-dependent manner,19 although the role of PECAM-1 in β-cat/TCF transcriptional regulation is not known. In this report, we describe the preferential activation of β-cat/TCF signaling in the atherosclerotic environment by hemodynamic shear stress, as well as its contribution to expression of the proatherogenic protein fibronectin. The overall goal of this work was to identify novel pathways involved in atherosclerosis development, inflammation, and shear stress that may provide valuable insight into the heterogeneous anatomic distribution of the disease, as well as providing new preventative or interventional opportunities.
Methods
Cell Culture
Primary human umbilical vein EC (HUVEC) were isolated as previously described20 and maintained in M199 (Lonza) with 10% fetal bovine serum (Gibco), 5 μg/mL EC growth supplement (Biomedical Technologies), 10 μg/mL heparin (Sigma-Aldrich), 2 mmol/L l-glutamine (Gibco), and 100 U penicillin/streptomycin (Invitrogen). Bovine aortic EC were maintained in Dulbecco's modified Eagle's medium (Gibco) supplemented with 10% fetal bovine serum, 2 mmol/L l-glutamine, and 100 U penicillin/streptomycin. β-cat-null murine EC (described here21) were maintained in MCDB-131 (Gibco) supplemented with 20% fetal bovine serum, 5 μg/mL EC growth supplement, 10 μg/mL heparin, 2 mmol/L l-glutamine, and 100 U penicillin/streptomycin.
Hemodynamic Shear Stress Application
Shear stress profiles measured from healthy human subjects17 were applied to cells using a cone and plate viscometer as previously described.20,22,23 Shear stress was applied for 24 hours except where indicated to assess the role of TCF/LEF signaling in a shear stress–adapted phenotype.22–24
Mice
Mouse studies were conducted with the approval of the University of Virginia Animal Care and Use Committee (ACUC#3597) and in accordance with the National Institutes of Health recommendation outlined in the Guide for the Care and Use of Laboratory Animals.
Male wild-type, apolipoprotein E (ApoE)−/−, and PECAM-1−/− ApoE−/− mice on a C57BL/6 (B6) background were maintained on a low-fat chow diet except where indicated. Studies were performed with a minimum of 3 mice in each condition. Heterozygous TOPGAL transgenic mice on a CD1 background (Jackson Laboratory) were crossed with B6 mice for 3 generations. To study TCF/LEF activity in atherosclerotic lesions, TOPGAL (CD1:B6) mice were crossed with ApoE−/− mice such that a minimum of 5 B6 backcrosses were completed. Mice were maintained on a low-fat chow diet and tissue was harvested between 12 and 20 weeks of age.
Statistics
Significance was determined through 2-tailed unpaired Student t test. Probability values <0.05 were considered statistically significant. All images and blots are representative of at least 3 independent experiments, and data are expressed as average±SEM.
Results
Regional Distribution and Activation of β-cat/TCF in Murine Endothelium in Vivo
The aortic arch is made up of geometrically opposed regions of relatively high and low shear stress. The greater curvature and descending aorta experience high, unidirectional shear stress and are resistant to atherosclerosis development (atheroprotective). Conversely, the lesser curvature of the aortic arch is exposed to lower, reversing shear stress (atheroprone) and, in hypercholesteremic animal models, tends to develop atherosclerosis.16 To assess the distribution of β-cat in environments of different atherosclerosis predisposition, tissue sections from wild-type B6 mice were examined (Figure 1A). In atheroprotective regions, β-cat was primarily localized to the cell-cell border, with modest cytosolic staining present in the greater curvature. In contrast, the atheroprone lesser curvature exhibited a strong nuclear fraction of β-cat.
Endothelial subcellular localization of β-cat depends on atherosclerotic susceptibility of the aortic region. A, En face staining of endothelial β-cat in different regions of the C57BL/6 mouse aorta. Images were acquired from the greater and lesser curvatures of the aortic arch and descending abdominal aorta. Images from greater curvature, lesser curvature, and descending aorta are representative of 3, 6, and 6 mice, respectively. B, En face staining of β-cat in atheroprotective (descending) and atheroprone (lesser curvature) regions of aortas of 8- to 13-week-old ApoE−/− mice. Arrows refer to exclusion of β-cat from the nuclei of EC of descending aorta. C, β-cat nuclear localization in endothelium of early atherosclerotic lesions in the greater (i to iii) and lesser (iv to vi) curvature of aortas of chow diet–fed 8- to 13-week-old ApoE−/− mice. To detect nuclear accumulation, aortic cross-sections were fluorescently stained and imaged for β-cat (red), nuclei (blue), and tissue autofluorescence (green) (i and iv). Single-channel images of nuclei (ii and v) and β-cat (iii and vi) reveal unique staining patterns. Arrows denote individual endothelial nuclei. Asterisks (*) denote a nascent atherosclerotic lesion. D, Quantitative analysis of endothelial nuclear β-cat in 22-week-old Western diet–fed ApoE−/− mice. Individual endothelial nuclei from 3 different mice (×, □, ▵) were separated into lesion-free (healthy) and lesion-burdened (lesion) groups and then assessed for relative nuclear β-cat expression. Horizontal dashed lines denote geometric mean of all nuclei for a given group. Error bars represent ±SD, *P<10−5.
To assess the distribution of β-cat in endothelium during atherosclerosis development, en face aortic sections from ApoE−/− mice were examined (Figure 1B). In atheroprotected regions, β-cat appears to be excluded from the nucleus (arrows). Atheroprone regions of ApoE−/− displayed higher β-cat present in the cytosol, though cells lacked clear nuclear definition. In cross-sections of early lesions (8 to 13 weeks of age), both nuclear β-cat and total levels of βcat were elevated compared with lesion-free areas (Figure 1C). In addition, nuclear β-cat correlated well with nuclear factor (NF)-κB—a hallmark of endothelial dysfunction (Supplemental Figure I, available online at http://atvb.ahajournals.org).25 Nuclear β-cat was also elevated in advanced atherosclerotic plaques, where endothelium overlying lesions exhibited 36% more nuclear β-cat than atherosclerosis-free regions in 22-week-old Western diet–fed ApoE−/− mice (P<0.05; Figure 1D).
Nuclear translocation of β-cat classically leads to TCF/LEF-dependent transcriptional activity. To directly assess the activity of TCF/LEF transcription factors in the murine aorta, transgene expression in reporter mice (TOPGAL) was measured. The expression of lacZ was greater in the atheroprone lesser curvature compared with the greater curvature (Figure 2A), suggesting that the atheroprone environment contributes to activation of TCF/LEF transcriptional activation. The activity of β-cat/TCF within developing atherosclerotic lesions was assessed in TOPGAL+/−/ApoE−/− mice (Figure 2B). LacZ expression was observed in endothelium superficial and adjacent to lesion development, which suggests that TCF/LEF-dependent transcription precedes lesion growth. Consistent with previous reports, fibronectin was strongly expressed in early atherosclerotic lesions (Figure 2C). Fibronectin expression and TCF/LEF activation exhibited parallel region-specific staining patterns. Cumulatively, these findings indicate that β-cat nuclear signaling preferentially occurs in regions predisposed to atherosclerosis.
Preferential TCF/LEF activation in the atherosclerotic prone aorta. A, En face analysis of TCF/LEF reporter TOPGAL transgenic mice. Mixed background CD1:B6 heterozygous transgenic reporter mice were assessed for the relative expression of lacZ within the atheroprotected greater and atheroprone lesser curvature of the aortic arch. Aortic rings were fluorescently stained for lacZ expression, and staining intensity was quantified. (n=3, *P<0.05). B, TCF/LEF activation in the aorta during early atherosclerosis. LacZ expression was assessed in early atherosclerotic lesions of chow diet–fed 12- to 20-week-old TOPGAL+/−/ApoE−/− mice using immunohistochemistry in paraffin-embedded cross-sections. i, Representative aortic cross section of an early atherosclerotic lesion. ii and iii, Higher magnification images of lacZ expression. iv, No primary antibody staining control. v, TOPGAL-null ApoE−/− littermate stained for lacZ expression as a nonspecific control. Ab indicates antibody. C, Fibronectin expression correlates with TCF/LEF activity in early atherosclerosis. Sections adjacent to those presented in B were stained for fibronectin expression. ii and iii, Higher magnification images of fibronectin expression. iv, Isotype staining control.
Atheroprone Hemodynamics Promote β-cat/TCF Activation and Expression of Fibronectin
Because constitutive nuclear β-cat and TCF/LEF activities were observed in arterial regions that constantly experience low, reversing shear stress, we next tested relative activity of this pathway in response to hemodynamic forces. Cultured EC monolayers were subjected to human-derived atheroprone and atheroprotective shear stresses via a shear stress cell culture system (Figure 3A and Supplemental Figure II). Following shear stress exposure, levels of nuclear accumulation of β-cat were significantly increased by atheroprone compared with atheroprotective shear stress (Figure 3B). Furthermore, cells transfected with a TCF-responsive luciferase reporter plasmid exhibited significantly increased TCF activity when exposed to the atheroprone flow compared with atheroprotective flow (Figure 3C).
Shear stress differentially regulates β-cat nuclear localization and TCF/LEF transcriptional activation in EC. A, Schematic of human-derived shear stress profiles from the common carotid artery (protected) and internal carotid sinus (prone) measured previously.17 EC were exposed to these hemodynamic shear stress profiles for the indicated times by a cone and plate cell culture device described in the Methods section. B, Nuclear lysates from HUVEC exposed to either protected and prone shear stress for 24 hours were assessed relative β-cat levels by Western blot. TATA-binding protein (TBP) was used to ensure equal loading. *P<0.05, n=3. C, Bovine aortic EC transfected with TOP-Flash were used to assess TCF/LEF transcriptional activity under different shear stress profiles. Luciferase activity was measured after 16 hours of exposure to either protected or prone shear stress. *P<0.05, n=4 to 6.
To directly test the transcriptional capacity of β-cat/TCF signaling in response to arterial hemodynamic simulation, we focused on a small cohort of known TCF-dependent, atherosclerosis-related genes. To identify the requirement of β-cat/TCF signaling to gene expression, we analyzed mRNA transcripts from EC treated with adenovirus (Ad) containing either a dominant-negative (DN) TCF-4 lacking the N-terminal binding26 (Ad-DN-TCF4) or an empty cytomegalovirus promoter control and exposed to shear stress. Three candidate genes (CyclinD1, interleukin [IL]-8, and fibronectin) were identified as being both upregulated under atheroprone hemodynamics and inhibited by Ad-DN-TCF4 treatment (Figure 4A and Supplemental Figure III). Because of the critical role of fibronectin in endothelial biology, we focused on this target. Application of atheroprone hemodynamics increased expression of fibronectin mRNA by approximately 2-fold compared with atheroprotective flow (Figure 4A). Pretreatment of EC with Ad-DN-TCF4 significantly reduced expression of fibronectin under atheroprone flow. Protein analysis confirmed both the atheroprone hemodynamic-induced increase as well as the reduction in fibronectin protein levels by Ad-DN-TCF treatment (Figure 4B). This suggests a novel role for β-cat/TCF regulation of EC fibronectin in response to hemodynamic stimulation. To determine whether β-cat binds to the fibronectin promoter, we performed a chromatin immunoprecipitation assay. We observed a significant level of enrichment of the proximal fibronectin promoter when lysates were precipitated with a β-cat antibody (Figure 4C and Supplemental Figure IV) compared with mock control and compared with a more upstream genomic region. This finding confirms the specific interaction between β-cat and the fibronectin transcriptional regulatory region.
Fibronectin gene expression in EC exposed to prone shear stress depends on TCF/LEF transcription. A, HUVEC treated with either empty adenovirus (Ad-Empty) or adenovirus containing dominant negative N-terminal deleted TCF-4 (Ad-DN-TCF4) were exposed to atheroprotective or atheroprone shear stress for 24 hours. mRNA levels of fibronectin were assessed by reverse transcription polymerase chain reaction. Expression was normalized to β-2-microglobulin. *P<0.05, †P<0.01, n=4 to 6. B, HUVEC exposed to 24 hours of protected or prone shear stress in the presence of either Ad-Empty or Ad-DN-TCF4 were analyzed for fibronectin protein expression by Western blot. *P<0.05, †P<0.01, n=3 to 4. C, β-cat binds directly to the human fibronectin promoter in EC. Polymerase chain reaction amplification of the proximal human fibronectin promoter was performed following pull-down with an anti-β-cat antibody or rabbit IgG (mock) control. Upstream control refers to polymerase chain reaction amplification of a region 2 kb upstream of the proximal fibronectin promoter. *P<0.05, n=6. D, β-cat staining localizes to the cell-cell border in wild-type murine EC (MEC) (left) but is absent in knockout cells (middle). Knockout cells reconstituted with Xβ-cat-Eng and stained with anti-Engrailed antibody showed localization to the cell-cell border (arrows), demonstrating proper incorporation to the adherens junction. E, Murine EC were exposed to 24 hours of atheroprone hemodynamics, and fibronectin protein levels were assessed. *P<0.05, n=3. F, During shear stress application, HUVEC were treated with dimethyl sulfoxide (DMSO) or 5 μmol/L compounds ICG-001 or IQ-1 to inhibit β-cat/CREB-binding protein (CBP) and β-cat p300 interactions, respectively. Fibronectin mRNA and protein levels were assessed by polymerase chain reaction and Western blot. *P<0.05, n=3 to 7. G, HUVEC were pretreated with either Ad-Empty or Ad-DN-TCF4 in combination with an NF-κB reporter and exposed to atheroprone shear stress for 24 hours. Following shear stress exposure, lysates were assessed for luciferase activity. †P<0.01, n=4.
To confirm the requirement of β-cat in fibronectin expression, we next interrogated the expression of fibronectin in β-cat knockout murine EC. Cells were reconstituted with Xenopus β-cat in the presence or absence of a Drosophila-derived engrailed repressor domain fused to the C terminus (Xβ-cat-Eng) (Figure 4D). This construct has been shown to effectively inhibit β-cat/TCF transcriptional activity.27 Atheroprone hemodynamic-induced expression of fibronectin was rescued in knockout cells reconstituted with Xβ-cat, but not in cells expressing Xβ-cat-Eng (Figure 4E), providing direct evidence for the transcriptional requirement of β-cat in fibronectin expression. Thus, the requirement of β-cat/TCF signaling in shear stress–induced fibronectin expression was substantiated by loss- and gain-of-function experiments.
To achieve maximal activation, β-cat must recruit histone acetyl transferases, including CREB-binding protein (CBP)28 and p300,29 to the transcription complex. Human EC were exposed to specific inhibitors of β-cat/CBP and β-cat/p300 interactions (ICG-00130–33 and IQ-134 respectively). Inhibition of β-cat/CBP interactions inhibited both fibronectin gene and protein expression (Figure 4F). Pharmacological inhibition of β-cat/p300 had no statistically significant effect, though we cannot eliminate the possibility of β-cat/p300 regulation of fibronectin expression. This suggests that recruitment of CBP to the β-cat/TCF complex is required for expression of fibronectin in EC induced by atheroprone hemodynamics.
DN-TCF4 Inhibits Shear Stress–Induced EC Inflammation
The transcription factor complex NF-κB represents a major inflammatory mediator in EC and atherosclerosis. Atheroprone shear stress enhances NF-κB activity in vitro to prime EC toward a proinflammatory phenotype.13,25 Here we wanted to test the hypothesis that downstream activation of β-cat/TCF contributes the “primed” NF-κB activation in response to atheroprone flow. Infection with Ad-DN-TCF4 reduced atheroprone flow-stimulated NF-κB activity by 70% (Figure 4G), supporting the physiological importance of β-cat/TCF signaling in the atheroprone environment.
PECAM-1 Is Required for Constitutive β-cat Nuclear Localization
Having established that activation of β-cat/TCF signaling occurs in response to hemodynamics, we next interrogated the importance of a critical shear stress–sensing protein PECAM-1. Immunostained aortic sections from young (8-week-old) PECAM-1−/−ApoE−/− mice exhibited no nuclear localized β-cat compared with age-matched ApoE−/− mice (Figure 5A). In addition, 8- to 13-week-old PECAM-1−/− ApoE−/− mice, whose ApoE−/− counterparts exhibited early lesion development, showed no nuclear β-cat staining in atheroprone regions (Figure 5B). Together, these observations point toward a novel regulatory role for PECAM-1 in the constitutive regional activation of β-cat nuclear translocation.
Regulation of nuclear β-cat localization and GSK-3 activity by platelet endothelial cell adhesion molecule-1 (PECAM-1) preceding and during atherosclerosis. A, En face fluorescent staining of β-cat and PECAM-1 in atheroprone regions of chow diet–fed ApoE−/− and PECAM-1−/−ApoE−/− aortas before atherosclerotic plaque development (8 weeks). B, Cross-sections of the lesser curvature of aortas of 8- to 13-week-old chow diet–fed PECAM-1−/−ApoE−/− mice. Tissue was immune-stained and imaged for β-cat (red), nuclei (blue), and tissue autofluorescence (green) (i). Single-channel images of nuclei (ii) and β-cat (iii) reveal decreased nuclei/β-cat colocalization (compare with Figure 1B). Arrows denote individual EC. C, HUVEC treated with either scrambled small interfering RNA (siCtl) or small interfering RNA targeted against PECAM-1 (siPECAM-1) were exposed to 24 hours of prone shear stress and assessed for relative levels of pS-GSK-3β by Western blot. **P<0.001, n=4 to 6. D, Aortas of Western diet–fed 22-week-old ApoE−/− (i to iii) and PECAM-1−/−ApoE−/− (v to vii) mice were stained for pS-GSK-3β. i and v, Low-magnification images of IgG control-stained (left) and anti-pS-GSK-3β-stained (right) aortic cross-sections. Expression was assessed in lesion-burdened (ii and vi) and non–lesion-burdened (ie, healthy) (iii and vii) endothelium. A histogram of pixel intensities of lesion and healthy EC pS-GSK-3β expression for ApoE−/− (iv) and PECAM-1−/−ApoE−/− (viii) revealed increased pS-GSK-3β levels in atherosclerotic plaques of ApoE−/− but not in PECAM-1−/−ApoE−/− plaques (the rightward shift of pS-GSK-3β intensity in lesion-burdened EC in ApoE−/− was absent in PECAM-1−/−ApoE−/−).
Shear Stress–Induced Inhibition of Endothelial GSK-3β Depends on PECAM-1
The presence of nuclear β-cat and active TCF/LEF in endothelium of atherosclerotic lesions suggests inhibition of the β-cat degradation pathway. Following exposure to atheroprone hemodynamics, phospho-serine-GSK-3β (pS-GSK-3β) levels were found to depend on PECAM-1 expression (Figure 5C).
Atherosclerotic Lesions Exhibit Elevated GSK-3β Inhibition, Which Depends on PECAM-1
We next assessed the relative abundance of inactivated GSK-3β in atherosclerotic lesions. pS-GSK-3β levels were elevated at sites of atherosclerotic lesion in ApoE−/− mice (Figure 5D, ii versus iii). In PECAM-1−/−ApoE−/− aortas, pS-GSK-3β levels were homogenous around the aortic circumference, regardless of lesion burden (Figure 5D, vi versus vii). Thus, in PECAM-1+/+, the endothelium exists in 2 distinct populations of pS-GSK-3β expression, whereas genetic deletion of PECAM-1 imparts uniformity along the vessel lumen (Figure 5D, iv versus viii). pS-GSK-3β levels correlate with both nuclear β-cat colocalization (Supplemental Figure II), as well as fibronectin expression. This implicates PECAM-1 as a critical regulator of pS-GSK-3β in atherosclerotic endothelium.
Discussion
This study identifies for the first time the activation of β-cat/TCF signaling in the endothelium before and during early atherogenesis. Our data suggest that atheroprone shear stress serves as a potent activator of endothelial β-cat/TCF signaling through a PECAM-1/GSK-3β-dependent mechanism and that this in turn drives transcription of fibronectin, a critical regulator of endothelial phenotype.
Activation of β-cat/TCF signaling is known to occur in response to a diverse set of cellular cues. Classically, initiation of TCF activity occurs through canonical Wnt signaling, where soluble Wnt factors bind to the Frizzled family receptors, leading to repression of the GSK-3β/Axin/adenomatous polyposis coli complex. Nuclear translocation of β-cat in EC by canonical Wnt signaling within the vascular wall is conceivable, given that they express multiple Frizzled receptors.35 However, the presence of canonical Wnt ligands in the vessel wall during atherosclerosis is unknown. A recent study points to the presence of Wnt pathway antagonist Dickkopf-1 in atherosclerotic plaques.36 In addition, the noncanonical Wnt-5a was found to be abundantly expressed in plaques37 and may inhibit canonical Wnt signaling.38 In addition to shear stress, other atherogenic stimuli induce activation of β-cat/TCF signaling in EC. IL-1β alone or in combination with tobacco smoke extract activates TCF/LEF through AKT/GSK-3β.39 Tumor necrosis factor-α drives paracrine Wnt signaling to promote osteogenesis in arterial smooth muscle cells.4 In addition to IL-1β, lipopolysaccharide also induced β-cat nuclear accumulation (Supplemental Figures V and VI), suggesting that activation of this pathway may represent a generic inflammatory response. In the context of the present study, the activation of TCF/LEF likely occurs as a result of a balance between promoting and antagonizing signals.
Our findings support the idea that PECAM-1 is a critical regulator of nuclear accumulation of β-cat in EC within developing lesions. Previous work documents a role for PECAM-1 in regulating β-cat localization in the absence of shear stress and atherosclerotic burden. However, divergent responses were reported depending on cell type,40,41 suggesting that this process depends on cell-specific factors, including SHP-2 activity. In the context of atherosclerosis development, PECAM-1 promotes nuclear accumulation of endothelial β-cat, consistent with its function in regulating shear stress–dependent endothelial inflammation. This likely occurs via regulation of GSK-3β phosphorylation, which is both shear stress and PECAM-1 dependent.19,42 Small interfering RNA–mediated knockdown of PECAM-1 impaired pS-GSK-3β levels, revealing a novel role for PECAM-1 in atheroprone shear stress–adapted EC and a potential mechanism for increased β-cat/TCF activity in atherosclerotic endothelium. Our data support the idea that prolonged exposure to atheroprone hemodynamics induces sustained GSK-3β inactivation. The onset of relatively high levels of shear stress induces an acute inhibition of GSK-3β,19 which may represent an early adaptation to shear stress. This is conceptually consistent with activation of NF-κB13, Rac1,43 extracellular signal–regulated kinase,42 p38,44 and AKT45 signaling in response to acute shear stress exposure, which is returned to baseline levels within minutes to hours. Interestingly, inhibition of GSK-3β is substrate dependent, where the RGD-rich matrix promotes GSK-3β phosphorylation compared with collagen substrates,42 suggesting that the existence of GSK-3β/fibronectin feedback that amplifies β-cat/TCF signaling. Indeed, β-cat, itself a gene target of β-cat/TCF, is differentially regulated in relation to shear stress magnitudes in porcine iliac arteries, suggesting a complex role for hemodynamics in the regulation of this pathway.46
Before this study, the consequences of endothelial β-cat/TCF activation on lesion biology were not known. We have shown that DN-TCF4 inhibits NF-κB in the atheroprone hemodynamic environment, suggesting that β-cat/TCF activity contributes to endothelial inflammation, thus promoting lesion advancement. This proinflammatory function of endothelial β-cat/TCF activity is likely related to fibronectin expression. Small interfering RNA-mediated knockdown of fibronectin is sufficient to decrease NF-κB activity in the atheroprone hemodynamic environment, which is rescued by treatment with exogenous fibronectin.10
Fibronectin expression, shown here to require CBP/β-cat/TCF under atheroprone shear stress, exerts multiple proatherogenic effects. Shear stress activation of NF-κB and JUN N-terminal kinase is enhanced on cells plated on fibronectin matrices.11,47 Fibronectin also contributes to shear stress–induced endothelial barrier dysfunction via activation of p21-activated kinase.48 The mechanism by which β-cat/TCF signaling drives transcription of fibronectin gene expression is not completely known. A consensus TCF/LEF binding site was identified in the Xenopus fibronectin promoter7; however, our analysis of the human fibronectin promoter revealed no putative proximal TCF/LEF consensus binding site. β-cat has been shown to bind to the fibronectin promoter independently of TCF/LEF in SW480 cells.49 In this regard, the CBP/β-cat/TCF complex may be critical for fibronectin expression, independently of TCF/DNA binding. We confirmed via chromatin immunoprecipitation assay that β-cat binds to the proximal fibronectin promoter in EC. Together, this supports that regulation of fibronectin by β-cat may occur via both direct TCF-independent and indirect TCF-dependent transcriptional regulators.
In addition to fibronectin, more than 50 genes have been identified as being TCF/LEF dependent.50,51 Among these are several atherosclerosis-associated genes, including CyclinD152 and IL-8,53 whose transcript levels were elevated by atheroprone hemodynamics and significantly inhibited by DN-TCF-4 treatment, suggesting that β-cat/TCF activity contributes broadly to endothelial gene expression in atheroprone regions. It is appreciated that endothelial turnover at atherogenic sites is significantly elevated. It is conceivable that β-cat/TCF-dependent CyclinD1 expression plays a role in this process.54 Furthermore, although elevated TCF/LEF reporter activity in atherosclerotic lesions was isolated to the endothelium and subendothelial foam cells, we cannot exclude that activation of this pathway in other cells contributes to lesion development. We observed β-catenin levels in smooth muscle cells, as well as several circulating cells that were likely monocytes.
Collectively, this study identifies a novel pathway activated in the early atheroprone environment, as well as revealing the regulation of a key player in arterial remodeling during atherosclerosis development. We demonstrate that β-cat nuclear localization and TCF/LEF transcriptional activation in the aortic endothelium occurs in response to atheroprone hemodynamic stimulation and precedes lesion development or advancement. This phenomenon may have diverse effects on atherosclerotic lesion biology in addition to its role in the regulation of the proinflammatory molecule fibronectin, and future study is warranted to identify other β-cat/TCF signaling targets in atherosclerosis.
Sources of Funding
This study was performed with support from National Institutes of Health Grant R01-HL082836 and R01-HL080956 (to B.R.B.), R01-HL081682 (to B.R.W.). Dr Gelfand was supported by the National Institutes of Health Training Grant 5T32HL007284.
Disclosures
None.
Acknowledgments
We acknowledge Dr Ryan Feaver, John Sanders, and Dr Michael Simmers for technical advice.
- Received November 30, 2010.
- Accepted March 13, 2011.
- © 2011 American Heart Association, Inc.
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